CRISPR/Cas-related methods and compositions for treating Usher syndrome and retinitis pigmentosa

Abstract

CRISPR/Cas-related compositions and methods for treatment of Usher Syndrome and/or Retinitis Pigmentosa are disclosed herein.

What is claimed is: 
     
       1. A method of altering a cell comprising contacting the cell with:
 (a) a first gRNA comprising a first targeting domain which is complementary with a first target domain from the USH2A gene, wherein the first targeting domain is configured to provide a first cleavage event selected from a first double strand break and a first single strand break in a region of the USH2A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a nucleic acid sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401; and 
 (b) a Cas9 molecule, 
 wherein an NHEJ-mediated indel is generated by the first break, resulting in a deletion in the USH2A gene. 
 
     
     
       2. The method of  claim 1 , wherein the cell is from a subject suffering from or likely to develop Usher Syndrome or retinitis pigmentosa-39. 
     
     
       3. The method of  claim 1 , wherein the cell is from a subject having a mutation in the USH2A gene. 
     
     
       4. The method of  claim 1 , wherein the cell is a photoreceptor cell. 
     
     
       5. The method of  claim 1 , wherein the contacting is performed ex vivo. 
     
     
       6. The method of  claim 5 , wherein the contacted cell is returned to the subject's body. 
     
     
       7. The method of  claim 1 , wherein the contacting is performed in vivo. 
     
     
       8. The method of  claim 1 , wherein the contacting comprises contacting the cell with a nucleic acid comprising a sequence encoding (a) the first gRNA. 
     
     
       9. The method of  claim 1 , further comprising contacting the cell with (c) a second gRNA. 
     
     
       10. The method of  claim 9 , wherein the second gRNA comprises a targeting domain which is complementary with a target domain from the USH2A gene, wherein the second targeting domain is configured to provide a second cleavage event selected from a second double strand break and a second single strand break, within 200 nucleotides of the target position of the guanine deletion at nucleotide position 2299 (2299delG) in the USH2A gene. 
     
     
       11. The method of  claim 10 , wherein the second cleavage event is in a region of the USH2A gene which is complementary to a sequence that is the same as, or differs by no more than 3 nucleotides from, a nucleic acid sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401. 
     
     
       12. A method of altering a cell comprising contacting the cell with:
 (a) a first gRNA comprising a first targeting domain which is complementary with a first target domain from the USH2A gene, the first targeting domain comprising a sequence that is the same as, or differs by no more than 3 nucleotides from, a first targeting domain sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401, 
 the first targeting domain configured to provide a cleavage event selected from a first double strand break and a first single strand break, in the USH2A gene; and 
 (b) a Cas9 molecule. 
 
     
     
       13. The method of  claim 12 , wherein the cell is from a subject suffering from or likely to develop Usher Syndrome or retinitis pigmentosa-39. 
     
     
       14. The method of  claim 12 , wherein the cell is from a subject having a mutation in the USH2A gene. 
     
     
       15. The method of  claim 12 , wherein the cell is a photoreceptor cell. 
     
     
       16. The method of  claim 12 , wherein the contacting is performed ex vivo. 
     
     
       17. The method of  claim 14 , wherein the contacted cell is returned to the subject's body. 
     
     
       18. The method of  claim 12 , wherein the contacting is performed in vivo. 
     
     
       19. The method of  claim 12 , wherein the contacting comprises contacting the cell with a nucleic acid comprising a sequence encoding (a) the first gRNA. 
     
     
       20. The method of  claim 12 , further comprising contacting the cell with (c) a second gRNA. 
     
     
       21. The method of  claim 20 , wherein the second gRNA comprises a targeting domain which is complementary with a target domain from the USH2A gene, wherein the second targeting domain is configured to provide a cleavage event selected from a second double strand break and a second single strand break, within 200 nucleotides of the target position of the guanine deletion at nucleotide position 2299 (2299delG) in the USH2A gene. 
     
     
       22. The method of  claim 20 , wherein the second targeting domain comprises a sequence that is the same as, or differs by no more than 3 nucleotides from, a second targeting domain sequence selected from the group consisting of SEQ ID NO:635, SEQ ID NO:392, SEQ ID NO:393, SEQ ID NO:394, SEQ ID NO:395, SEQ ID NO:396, SEQ ID NO:397, SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:400, and SEQ ID NO:401.